The proposed studies are designed to characterize the properties and behavior of a hormone- and cAMP-responsive steroidogenesis activator polypeptide (SAP) previously identified by us in the rat adrenal cortex and gonads. This cycloheximide-sensitive 3215- kDal factor, recently isolated from a rat Leydig cell tumor and sequenced, is believed to be the long-sought "labile protein" implicated in the acute regulation of mitochondrial cholesterol side-chain cleavage in steroid hormone-producing tissues. Using dispersed cells from the inner zones of the adrenal cortex and antisera we have raised to SAP, we will investigate the time- and concentration-dependent changes in the intracellular content of SAP in response to ACTH, 8-Br-cAMP, and inhibitors of protein translation and transcription. Details of the mechanism by which cAMP promotes a rise in the cell content of SAP will be investigated, with particular attention focused on a model invoking proteolytic generation of the functional peptide from an inactive precursor. Studies are also designed to assess the metabolic fate of the polypeptide. The potential trophic influence of ACTH on SAP at the transcriptional level will be addressed by monitoring changes in the cellular content of SAP mRNA. Insight into the mechanism by which SAP activates cholesterol side-chain cleavage will be had by testing for various properties which might sensibly be attributed to it. These include the capacity to promote cholesterol interaction with cytochrome P-450 side chain cleavage, modulate membrane cholesterol redistribution, influence the physical relationship between inner and outer membranes, bind calcium, or allosterically regulate some mitochondrial enzyme or structural protein associated with the cholesterol side-chain cleavage reaction. In addition to the adrenocortical studies, we will investigate several gonadal systems in which the role of SAP may prove especially interesting--the superovulated ovary, the "early lesion" of gonadotropin-induced heterologous desensitization in the testis, and the rat R2C Leydig cell tumor, in which cycloheximide- sensitive steroidogenesis proceeds in a constitutive manner. These investigations have the potential for substantially enhancing our understanding of the way in which steroidogenesis is regulated at a very fundamental level.